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| = Build Binary Reference Genome and Word Index = | | = Build Binary Reference Genome and Word Index = |
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− | First, build a binary version of the genome reference sequence as nucleotides (option: --createReference). Suppose that NCBI36.fa is a FASTA file which contains nucleotide sequences for all chromosomes. | + | First, build a binary version of the genome reference sequence as nucleotides (option: --createReference). Suppose that NCBI36.fa is a FASTA file which contains the nucleotide sequences for all chromosomes. |
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| The command to invoke is: | | The command to invoke is: |
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| karma --createReference --reference NCBI36.fa | | karma --createReference --reference NCBI36.fa |
| <br> | | <br> |
− | (To let KARMA map nucleotide space reads, one would use instead ''--createIndex'' to create both a binary sequence and the word index files.)<br> | + | (To let KARMA map nucleotide space reads, one would use instead ''--createIndex'' to create both a packed binary sequence file and the word index files.)<br> |
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| Second, one also needs to build color space versions of both the genome reference sequence (option: --createReference) and the word index files (option: --createIndex). The same nucleotide FASTA file is used. However, to avoid naming conflicts among the resulting binary files, we suggest appending "CS" to the base file name for clarity. The command to invoke is:<br> | | Second, one also needs to build color space versions of both the genome reference sequence (option: --createReference) and the word index files (option: --createIndex). The same nucleotide FASTA file is used. However, to avoid naming conflicts among the resulting binary files, we suggest appending "CS" to the base file name for clarity. The command to invoke is:<br> |
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| = Map Color Space Reads = | | = Map Color Space Reads = |
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− | KARMA expects valid color space FASTQ files as input. We often use the suffix .csfastq to distinguish these from nucleotide space reads. For a .csfastq file of single end color space reads named single.csfastq, invoke the command:<br> | + | KARMA expects valid color space FASTQ files as input. We often use the suffix .csfastq to distinguish these from nucleotide space reads. With a .csfastq file of single end color space reads named single.csfastq, invoke the command:<br> |
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| karma --reference NCBI36.fa --csReference NCBI36CS.fa --colorSpace single.csfastq | | karma --reference NCBI36.fa --csReference NCBI36CS.fa --colorSpace single.csfastq |
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− | This command line specifies both the nucleotide and color space reference sequences (and the word indexes, invisibly). The output will be written to a file in .sam format named "single.sam".<br> | + | This command line specifies both the nucleotide and color space reference sequences (and the word indexes, invisibly). The output will be written to a file in .sam format named "single.sam" derived from the .fastq file name.<br> |
| <br> | | <br> |
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− | Multiple input files are also acceptable, e.g.<br> | + | Multiple input files are also acceptable and will produce multiple .sam output files, e.g.<br> |
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| karma --reference NCBI36.fa --csReference NCBI36CS.fa --colorSpace \ | | karma --reference NCBI36.fa --csReference NCBI36CS.fa --colorSpace \ |
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| --pairedReads pair.1.csfastq pair.2.csfastq | | --pairedReads pair.1.csfastq pair.2.csfastq |
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− | The mapping results will be stored in a .sam file named "pair.1.sam", which contains reads from both files. If multiple paired end read files are specified on the command line, KARMA will pair the 1st and 2nd files, 3rd and 4th files and etc.<br> | + | The mapping results will be stored in a .sam file named "pair.1.sam", which contains reads from both files. If multiple paired end read files are specified on the command line, KARMA will pair the 1st and 2nd files, 3rd and 4th files, etc. and write output files "pair.1.sam", "pair.3.sam", etc.<br> |
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| karma --reference NCBI36.fa --csReference NCBI36CS.fa --colorSpace \ | | karma --reference NCBI36.fa --csReference NCBI36CS.fa --colorSpace \ |
| --pairedReads pair.1.csfastq pair.2.csfastq pair.3.csfastq pair.4.csfastq | | --pairedReads pair.1.csfastq pair.2.csfastq pair.3.csfastq pair.4.csfastq |
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− | = <br> Additional Information<br> = | + | = Additional Information = |
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| == Input file requirement == | | == Input file requirement == |
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| == Minimum read length requirement == | | == Minimum read length requirement == |
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− | Keep in mind that KARMA requires color space reads that are at least twice as long as the index word size plus two (including the leading primer base). (For nucleotide space, the minimum read length is twice the word size.) For example, KARMA uses an index word size of 15 by default, so it will only map color space reads that are 32 base pairs or longer.<br> | + | Keep in mind that KARMA requires color space reads that are at least twice as long as the index word size plus two (including the leading primer base). (For nucleotide space, the minimum read length is twice the word size.) For example, KARMA uses an index word size of 15 by default, so it will only map color space reads that are 32 colors or longer (including the primer base).<br> |
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| == Auxiliary tools == | | == Auxiliary tools == |
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− | The ABI SOLiD platform generates separate FASTA and quality files named XXX.csfasta and XXX\_QV.qual. We provide a script ''solid2csfastq.py'' which converts these into a single color space FASTQ file named XXX.csfastq. We believe that a single color space FASTQ file simplifies post processing.<br> | + | The ABI SOLiD platform generates separate FASTA and quality files named XXX.csfasta and XXX_QV.qual. We provide a script ''solid2csfastq.py'' which converts these into a single color space FASTQ file named XXX.csfastq. We believe that a single color space FASTQ file simplifies post processing.<br> |
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| == Choose an appropriate size for word index == | | == Choose an appropriate size for word index == |